Non-aflatoxigenic aspergillus parasiticus strains and their use in controlling aflatoxin contamination

ABSTRACT

A process for biologically controlling the preharvest accumulation of aflatoxin in soil-borne crops. Non-aflatoxigenic strains of Aspergillus parasiticus having all of the relevant identifying characteristics of NRRL 18786 and NRRL 18991 are shown to inhibit aflatoxin production by native toxigenic strains of Aspergillus flavus or Aspergillus parasiticus in the soil environment.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for the preharvest control ofaflatoxin in crops. This is accomplished by inoculating either the cropor the soil in which it is grown with non-aflatoxigenic strains ofAspergillus parasiticus (A. parasiticus), deposited and designated asNRRL 18786 and NRRL 18991.

2. Description of the Prior Art

Aflatoxins are potent hepatotoxic, carcinogenic compounds produced byfungi, particularly Aspergillus flavus (A. flavus) and A. parasiticus[Cast, (1989) Mycotoxins: economic and health risks. Report 116. Councilfor Agricultural Science and Technology). When these fungi invade andgrow in agricultural commodities such as peanuts, corn, and cottonseed,the resulting contamination with the aflatoxins often makes thecommodity unfit for consumption. They are a serious threat to humans andanimals [Cast, Counc. Agric. Sci. Technol. Rep., Vol. 80, (1979), Ames,IA., 56 pp.]. The four naturally-occurring aflatoxins are designatedB₁,B₂,G₁, and G₂ and will hereafter be collectively referred to asaflatoxin.

The United States peanut industry has identified aflatoxin contaminationof peanuts as the number one problem for which a solution is needed[Consensus Report of the National Peanut Council Quality Task Force(1987) National Peanut Council].

Because peanuts are used primarily for food, strict regulatory limitsfor the amount of aflatoxin allowable in finished peanut products havebeen established. Although the U.S. Food and Drug Administration (FDA)has an action level of 20 parts per billion (ppb) of total aflatoxins infinished products, several states are considering much stricter limitsfor aflatoxin in food. For this reason the U.S. peanut industry has agoal to ensure the delivery of aflatoxin-free peanut products by theyear 2000.

Although aflatoxin contamination of peanuts can occur during postharvestcuring and storage, the most significant source of contamination isusually preharvest contamination, which occurs during periods oflate-season drought stress as peanuts are maturing. The only knownmethod for controlling preharvest aflatoxin contamination in peanuts isirrigation [Cole, R. J. (1982), Dev. Ind. Microbiol. Vol. 23, pp.229-236; Cole, R. J., Aflatoxin Contamination of Groundnut: Proceedingsof the International Workshop, Oct. 6-9, 1987, ICRISAT Center, India],an option that is unavailable to the majority of peanut growers.

It has previously been found that co-cultivation of either A.parasiticus or A. flavus with species of Penicillia reduce levels ofaflatoxin production while co-cultivation with Fusaria had no sucheffect [Ehrlich, et al., Experientia, Vol. 41, pp. 691-693, (1985)].These tests did not involve the use of a soil environment.Co-cultivation with A. niger completely eliminated the production ofaflatoxin by a producer culture of A. flavus [Wicklow, et al.,Phytopathology, Vol. 70, pp. 761-764, (1980)]. This testing was doneunder laboratory controlled conditions in which the food source involvedsterilized corn kernels.

SUMMARY OF THE INVENTION

It has now been discovered that a known strain of Aspergillusparasiticus (NRRL 18991) is non-aflatoxigenic and competes with nativetoxigenic strains of A. parasiticus and A. flavus under fieldconditions, causing a reduction in aflatoxin production and subsequentcrop contamination. It has further been discovered that anon-aflatoxigenic mutant (NRRL 18786) selected following UV mutagenesisof Aspergillus parasiticus NRRL 18991, unlike its parent, fails toproduce O-methylsterigmatocystin (OMS) which is mutagenic andpotentially carcinogenic; yet still retains the ability to reduce theaflatoxin production of native strains through biocompetition.

In accordance with this discovery, it is an object of the invention tobiologically control the preharvest accumulation of aflatoxin insoil-borne crops.

A further object of the invention is to define a novel mutant species ofAspergillus which is non-toxigenic.

Other objects and advantages of this invention will become readilyapparent from the ensuing description.

DETAILED DESCRIPTION OF THE INVENTION

The underlying theory of the instant invention is that addition of ahighly competitive, non-toxigenic strain of A. parasiticus(biocompetitive agent) to soil would result in lower concentrations ofaflatoxin in peanuts. The rationale is that the biocompetitive agentwould dominate the soil microflora and prevent the buildup ofaflatoxin-producing strains of A. flavus/parasiticus that normallyoccurs during late-season drought. Through biological competition, thetoxigenic strains found naturally in soil would be replaced by anon-toxigenic strain added to the soil. Therefore, peanuts subjected tolate-season drought stress would be invaded predominately by thebiocompetitive agent, which is unable to produce aflatoxin. The fungiuseful in the instant invention are strains possessing the identifyingcharacteristics of A. parasiticus (NRRL 18991 and NRRL 18786). A.parasiticus NRRL 18991 isolated from the environmental control plots atthe National Peanut Research Laboratory (NPRL) in 1980 and subsequentlyshown not to produce aflatoxin [Dorner, J. W. (1984), Mycopathologia,Vol. 87, pp. 13-15]. At that time, it was the only naturally-occurringstrain of A. parasiticus known that did not produce aflatoxin. Thisparticular strain was selected for use in the instant invention becauseit met several heretofore unrecognized criteria. First, for an organismto be successful as a biocompetitive agent for aflatoxin control, itshould occupy the same ecological niche as A. flavus/parasiticus andcompete with the native strains under the environmental conditions thatlead to aflatoxin contamination; i.e., hot, dry peanut soil duringperiods of late-season drought stress. Second, it was found to be astable, non-producer of aflatoxin. Third, A. parasiticus was shown to bemore persistent in soil than A. flavus. [Cole, R. J. (1986), Appl.Environ. Microbiol. Vol. 52, pp. 1128-1131]. Fourth, this particularstrain was a prolific producer of sclerotia, which enhances itssurvivability and competitiveness in the soil. Although this strain(NRRL 18991) does not produce aflatoxin, it does produceO-methylsterigmatocystin (OMS), the immediate biosynthetic precursor toaflatoxin B₁. This fact provided the use of OMS as a chemical marker tomonitor the activity of the fungus in the soil and in peanuts.

The fact that A. parasiticus (NRRL 18991) accumulates OMS could make itunacceptable as a general-use biocompetitive agent because OMS ismutagenic and possesses the dihydrobisfuran moiety reportedlyresponsible for aflatoxin's carciogenicity [Cast (1989). Mycotoxins:economic and health risks. Report 116. Council for Agricultural Scienceand Technology, 91 pp.]. Because of these potential drawbacks, aUV-induced mutant of the NRRL 18991 strain was developed by growingcultures of the parent strain which were irradiated under short wave UVlight by the method of Bennett and Goldblatt [Bennett, J. W. and L. A.Goldblatt (1973), Sabouraudia, Vol. 11, pp. 235-241]. Surviving colonieswere isolated, grown on potato dextrose agar (PDA) slants and analyzedfor OMS and aflatoxin by adding 3 mL chloroform to the slant tube,vortexing for one minute, and filtering through microfiber filter paper.The filtrate was evaporated to dryness under nitrogen, redissolved in 50μL chloroform, and mixed. A 2 μL aliquot was spotted on a silica gel 60thin-layer chromatography (TLC) plate along with standards of aflatoxinand OMS, and plates were developed in a solvent system ofchloroform-acetone (93-7, v/v). Developed plates were viewed underlong-wave ultraviolet light before and after spraying with 50% ethanolicsulfuric acid. Aflatoxins were visualized as bluish or greenishfluorescent spots before spraying and as yellowish spots followingspraying. OMS was a blue fluorescent spot before spraying and anintensely yellow fluorescent spot after spraying. An acceptable mutantidentified as M52 (NRRL 18786) was subsequently found. NRRL 18786accumulates versicolorin A, another intermediate in aflatoxinbiosynthesis without producing detectable amounts of OMS.

A series of tests were carried out to determine the utility of theinstant disclosed strains in reducing aflatoxin contamination ofsoil-borne crops. The following examples are intended only to furtherillustrate the invention and are not intended to limit its scope whichis defined by the claims.

TEST PROCEDURE Soil Microflora Analysis

The relative number of colony forming units (CFU) of aflatoxin-producingstrains of A. flavus/parasiticus and the biocompetitive agents intreated and nontreated soils were determined at various times. Five soilsamples of approximately 250 g were taken to a depth of 5 cm at variouslocations in the plots. Each sample was well mixed and screened througha 20 mesh sieve. Fifteen g of screened soil was then added to 300 mL ofa sterile 0.2% agar solution in a 500 mL Erlenmeyer flask. Afterswirling, 10 0.5 mL portions (equivalent to 0.025 g of soil) were placedin the middle of petri plates containing 20 mL of the followingselective medium: 20 g agar; 10 g glucose; 5 g peptone; 30 g NaCl; 1 gK₂ HPO₄ ; 0.5 g MgSO₄ ; 6 mL of a botran solution (90 mL acetone, 30 mgbotran); 5 mL of an antibiotic solution (20 mL water, 0.2 gchlortetracycline HCl, 0.2 g streptomycin sulfate); 1 L distilled water(David M. Wilson, personal communication). A glass rod was placed on theagar as the plate was rotated to distribute the soil suspension over thesurface of the agar. For samples with colonies too numerous to count,appropriate dilutions of the soil suspension were made and samples werereplated. Plates were incubated for four days at 30° C. and A.flavus/parasiticus colonies were counted. CFU of these fungi per gram ofsoil were determined by multiplying the average number of colonies perplate by 40.

The A. flavus/parasiticus colonies were then tested to determine thepercentage that were aflatoxin producers, one of the biocompetitiveagents, or neither. Approximately 10% of the total colonies wererandomly chosen and transferred to PDA slants and incubated at 30° C.for 7 days. Each slant was analyzed for aflatoxin and the chemicalmarkers, OMS and versicolorin A, as described for the analysis of slantsof mutated colonies. The resulting percentages were then multiplied bythe total CFU per gram to determine the actual CFU per gram of eachtype.

EXAMPLE 1 First Crop Growing Season

Six rows, spaced 0.9 m apart, of cv. Florunner peanuts were grown in anenvironmental control plot (5.5 m×12.2 m) at the NpRL, Which providedthe late-season drought and soil temperature conditions that werepreviously determined to be optimum for preharvest aflatoxincontamination of peanuts. Cultural practices recommended by the GeorgiaCooperative Extension Service were used until 100 days after planting(DAP), at which time the peanuts received a final irrigation. The stressperiod was started at 107 DAP.

The biocompetitive agent (A. parasiticus, NRRL 18991) was grown in six2.8 L Fernbach flasks on liquid YES medium (15% sucrose; 5% mycologicalbroth, pH 4.8; 2% yeast extract) for 2 weeks at 27° C. The cultures werecombined and homogenized in 15 L of water plus 0.05% Tween 20 with anUltra-Turrax homogenizer. The homogenate was strained throughcheesecloth and applied over three of the six rows of peanuts at 32 DAPusing a garden sprinkler. A similar application was made 100 DAP, theday of the final irrigation. Each of the treated and nontreated rows wasdivided into sections so that four random samples of each were harvestedafter 23, 30, 37, and 44 days of stress. Samples were shelled with aFederal State Inspection Service sample sheller and sized intocommercial size categories (jumbo, medium, number 1, other-edible, andoil stock). Damaged and visibly molded kernels were removed from eachcategory and combined as a single category.

Jumbo, medium, number 1, and other-edible size peanuts were prepared foranalysis by grinding to pass a 20 mesh sieve. Aflatoxin analyses of eachcategory were carried out on 75% of the ground samples using the highperformance liquid chromatography (HPLC) method of Dorner and Cole. Alloil stock and damaged peanuts were analyzed for aflatoxin only. Theremaining 25% of the ground was analyzed for OMS with an HPLC methoddeveloped for this study. The ground peanut samples were extracted withchloroform, filtered through microfiber filter paper, evaporated to anoil on a rotary evaporator, and applied to a Florisil Sep PAK (WatersChromatography Division, Millipore Corp., Milford, Mass. 01757) incyclohexane. The Sep PAK was washed with 10 mL volumes of cyclohexane,cyclohexane-ethyl acetate (60-40, v/v), and OMS was eluted with 10 mL ofethyl acetate. The ethyl acetate fraction was evaporated to dryness andredissolved in 1 mL of ethyl acetate-hexane (85-15, v/v). Fifty μL wasinjected into an HPLC system consisting of a waters silica cartridge (5mm×10 cm) in a radial compression module, eluted with a mobile phase ofethyl acetate-hexane (85-15, v/v), and detected at 310 nm with a Watersmodel 490E programmable UV detector. Quantitation was achieved with aWaters model 730 data module which compared peak areas of samples toareas of OMS standard solutions.

Results of the First Crop Growing Season

Results of aflatoxin and OMS analyses are presented in Table 1. By 23stress days, aflatoxin concentrations were already high in the inediblepeanuts (other-edible, oil stock, and damaged peanuts). Aflatoxin hasbeen shown consistently to appear first and achieve higherconcentrations in these high risk categories. Therefore, in milling andprocessing operations, these peanuts are removed from peanuts destinedfor edible use, regardless of aflatoxin concentration. Aflatoxinconcentrations were unacceptably high in both treated and untreatededible peanuts (jumbo, medium, and number 1) from the 30 stress-daysampling. However, as the stress period continued aflatoxinconcentrations decreased in edible peanuts from soil that was treatedwith the biocompetitive agent to a level that is under the action levelset by the FDA. On the other hand, aflatoxin concentrations continued toincrease during the stress period in edible peanuts grown in soil thatwas not treated with the biocompetitive agent. Results of OMS analysesindicated that the biocompetitive agent was actively contaminatingpeanuts, but it had not excluded all wild, aflatoxigenic strains of A.flavus/parasiticus.

EXAMPLE 2 Second Crop Growing Season

Because of the positive results of the first crop growing season, thestudy was continued for a second crop growing season with severalmodifications. Preplant soil microflora analysis indicated that a largepopulation of the biocompetitive agent remained in the soil from theprevious year's study, including the area that was not treated with thebiocompetitive agent in the first crop growing season. Therefore, soilwas removed from one-half of the plot to a depth of 1 m and replacedwith new soil. A barrier was placed between the two halves of the plot,and the half containing the new soil served as a nontreated controlwhile the half with the 15 old soil was used to determine theeffectiveness of the biocompetitive agent for the second crop growingseason. No additional biocompetitive agent was added to the soil.

Florunner peanuts were grown and subjected to late-season drought stressas in the previous year. The final irrigation was applied 98 DAP, thestress period started 105 DAP, and all peanuts were harvested 154 DAPafter 49 days of stress. All peanuts from treated soil were analyzed foraflatoxin and OMS as in the first crop growing season, but only ediblepeanuts grown in the new soil were analyzed for aflatoxin.

In addition to the preplant soil microflora analysis, soil samples werealso taken at harvest to compare propagule levels of the biocompetitiveagent and wild strains of A. flavus/parasiticus with those determinedprior to planting.

Results of the Second Crop Growing Season

Results of the preplant and harvest soil microflora analyses arepresented in Table 2. Populations of both aflatoxin-producing strains ofA. flavus/parasiticus and the biocompetitive agent approximately doubledduring the season. However, the population of the biocompetitive agentfar outweighed that of wild-type aflatoxin producers. The finalpopulation of the biocompetitive agent was comparable to levels commonlyseen for A. flavus/parasiticus in peanut soils exposed to late-seasondrought stress (unpublished data). This demonstrated that a high degreeof replacement of toxigenic strains of A. flavus/parasiticus by thebiocompetitive agent occurred.

                  TABLE 1                                                         ______________________________________                                        Aflatoxin and O-methylsterigmatocystin (OMS) concentrations                   (ppb) in peanuts from soil treated and not treated with the                   biocompetitive agent in the first crop growing season.                        Stress Period        Aflatoxin      OMS                                       (days)    Treatment  Edible.sup.1                                                                           Inedible.sup.2                                                                        Edible                                  ______________________________________                                        23        Treated     4         577   15                                                Untreated   1         739    7                                      30        Treated    222      2,534   31                                                Untreated  97       4,775   41                                      37        Treated    19       11,783  120                                               Untreated  106      12,688   5                                      44        Treated    11       7,035   94                                                Untreated  531      21,692  81                                      ______________________________________                                         .sup.1 Values are the weighted average for jumbo, medium, and number 1        sizes.                                                                        .sup.2 Values are the weighted average for the otheredible, oil stock, an     damaged categories.                                                      

                  TABLE 2                                                         ______________________________________                                        Soil populations (CFU per gram) of aflatoxigenic strains of A.                flavus/parasiticus and biocompetitive agent prior to planting and at          harvest for the second crop growing season treated soil.                      Sampling  A. fIavus/parasiticus                                                                       Biocompetitive Agent                                  ______________________________________                                        Preplant  207            5,233                                                Harvest   442           10,925                                                ______________________________________                                    

Aflatoxin concentration in edible, treated peanuts in the second cropgrowing season were by far the lowest ever observed during nine years ofresearch using the environmental control plots (Table 3). Together,edible peanuts contained only 1 ppb of aflatoxin compared to 96 ppb inedible peanuts from untreated soil. By comparison, the OMS concentrationin edible peanuts from treated soil was 172 ppb, providing strongevidence that the biocompetitive agent had invaded peanuts andproliferated to a far greater extent than aflatoxigenic strains. Theinedible peanuts still had significant amounts of aflatoxin, but thesetoo were much lower than had been observed in previous crop growingseasons and were much lower than the amounts of OMS present.

                  TABLE 3                                                         ______________________________________                                        Second crop growing season aflatoxin and OMS concentrations                   (ppb) in peanuts from soil treated in the first crop growing season           with the biocompetitive agent and new, untreated soil. All peanuts            were subjected to 49 days of drought stress.                                  Category            Aflatoxin                                                                              OMS                                              ______________________________________                                        Treated Soil                                                                  Jumbo                0         45                                             Medium               0         238                                            Number 1             4         98                                             Edible weighted average                                                                            1         172                                            Other-edible         0       1,288                                            Oil stock            68      1,776                                            Damage              3,908    13,311                                           Inedible weighted average                                                                         515      2,945                                            Untreated Soil               N/A*                                             Edible               96                                                       ______________________________________                                         *Peanuts from untreated soil were not analyzed for OMS.                  

EXAMPLE 3 Third Crop Growing Season

The study of the second crop growing season was essentially repeated.Soil was again replaced in the untreated half of the plot and noadditional biocompetitive agent was added to the treated half. This soilwas last inoculated with the biocompetitive agent at the start of thefirst crop growing season.

Additional studies were conducted to determine the effectiveness of theM52 mutant from A. parasiticus NRRL 18991 as a biocompetitive agent.Four environmental control plots (5.5 m×6 m) were inoculated with twodifferent inoculum levels of the mutant in order to begin determiningthe effect of inoculum level on fungal soil populations and aflatoxincontamination. To determine the number of CFU in a Fernbach flask afterhomogenization with water and 0.05% Tween 20, serial dilutions of threeflasks were plated and the average determined to be approximately120×10⁹ CFU per flask. Therefore, it was decided to broadcast thehomogenized and cheesecloth-strained contents of five Fernbach flasks(600×10⁹ CFU) over each of two plots to serve as the high inoculumtreatments. Half the contents of one flask (60×10⁹ CFU) were broadcastover each of the other two plots to serve as low inoculum treatments.These plots were inoculated 57 days before peanuts were planted. The dayfollowing inoculation these plots were irrigated with 4 cm of water.

Peanuts for this and the continuing study were grown as in previousyears with final irrigation occurring 92 DAP, stress period beginning 99DAP, and harvesting 147 DAP (48 days of stress).

All peanuts were harvested and handled as previously described, exceptthat all peanuts were analyzed for aflatoxin only. Soil samples formicroflora analysis were collected from all plots on the day afterplanting, 99 DAP (beginning of stress period), and on the day ofharvest.

Results of the Third Crop Growing Season

Results of soil microflora analyses conducted during the third cropgrowing season appear in Table 4. As previously observed in new soil,wild strains of A. flavus/parasiticus were relatively low prior toplanting (40 propagules per gram) and increased dramatically by harvesttime (4386 ppg.). In the continuing study, populations of aflatoxinproducers and the biocompetitive agent remained fairly constant,indicating that this soil could not support a significant buildup ofeither type. In comparing the populations at harvest of the untreatedsoil with those of the soil from the continuing study, it is interestingthat the wild A. flavus propagules and biocompetitive agent propaguleswere essentially reversed in the two treatments. This shows thatintroduction of a relatively large fungal population did not result in afinal population that was higher than that of wild strains of A.flavus/parasiticus under these conditions.

                  TABLE 4                                                         ______________________________________                                        Fungal soil populations (CFU per gram) from the third crop                    growing season sampled prior to planting, 97 days after planting              (DAP), and at harvest.                                                                 Preplant 97 DAP     Harvest                                          Treatment  AF.sup.1                                                                             BA.sup.2                                                                              AF   BA    AF    BA                                 ______________________________________                                        Untreated   40    ND.sup.3                                                                              100  ND    4,386   255                              Ongoing    403    3,597   150   3,000                                                                                385  4,785                             High Inoculum                                                                            ND     21,200  ND   34,000                                                                                500 25,300                             (M52)                                                                         Low Inoculum                                                                             ND     3,800   ND    3,550                                                                              3,470  5,400                             (M52)                                                                         ______________________________________                                         .sup.1 Wild strains of aflatoxinproducing A. fIavus/parasiticus.              .sup.2 Biocompetitive agent. For the untreated soil and ongoing study,        this is the parent NRRL 18991 strain. For the high and low inoculum           treatments, this is the M52 mutant strain.                                    .sup.3 No CFU detected.                                                  

In the low and high inoculum treatments with the M52 mutant, much bettercontrol of wild A. flavus/parasiticus populations was achieved with thehigh inoculum treatment. This provides an indication that the amount ofinoculum is an important consideration in achieving control throughbiological competition.

Results of aflatoxin analyses for the third crop growing season studiesare presented in Table 5. A reduction in aflatoxin concentrations wasagain seen in peanuts grown in soils treated with a biocompetitiveagent. Significantly, the greatest effect was seen in the ediblepeanuts. The 40 ppb found in all edible peanuts from soil last treatedin the first crop growing season, which was a significant increase fromthe 1 ppb found in the second crop growing season, indicated that thebiocompetitive agent may have been losing its effectiveness after beingin the soil for three crop growing seasons. Over a ten-fold reduction inaflatoxin was found in edible peanuts grown in soil treated with thehigh inoculum level of the M52 mutant compared to untreated soil (17 vs.241 ppb).

                                      TABLE 5                                     __________________________________________________________________________    Third crop growing season aflatoxin concentrations (ppb) in peanuts from      untreated soil,                                                               soil treated with a biocompetitive agent in the first crop growing            season, (ongoing),                                                            and soil treated with high and low inoculum levels of the M52 mutant.                              Edible  Other                                                                             Oil      Inedible                            Treatment                                                                             Jumbo                                                                             Medium                                                                             No. 1                                                                             Weighted Avg.                                                                         Edible                                                                            Stock                                                                             Damage                                                                             Weighted Avg.                       __________________________________________________________________________    Untreated                                                                             232 132  393 241     474 3,774                                                                             107,158                                                                            26,876                              Ongoing  92 10   74  40      255 1,170                                                                             28,527                                                                             7,588                               High inoculum                                                                          6  22   15  17      179   90                                                                               6,786                                                                             2,022                               Low inoculum                                                                           8  25   38  29      302   40                                                                              21,875                                                                             7,071                               __________________________________________________________________________

Soil treated with the low inoculum level of the M52 mutant also resultedin a lower aflatoxin concentration in edible peanuts (29 ppb). Theresults from these treatments with the M52 mutant are more nearly likethose seen from the 37 and 44 stress day samplings taken in the firstcrop growing season , and coupled with the results from the second cropgrowing season may indicate that maximum control is achieved in the cropgrowing season following inoculation.

There has been provided in accordance with the present invention, abiocontrol agent, compositions, and methods for the prevention and/orcontrol of aflatoxin contamination in agricultural commodities. It canbe seen from the preferred embodiments that many variations andalternatives may be practiced without departing from the spirit andscope of the invention. It is intended that the scope of this inventionincludes such variations and alternatives.

It is envisioned that any system for biocontrol delivery, known to theskilled artisan, can be used for the administration of non-aflatoxigenicA. parasiticus strains to agricultural crops of the soil in which theyare grown. Additionally, carrier agents for biocontrol can be inertcompounds or compositions including stabilizers and preservatives knownin the art.

We claim:
 1. A method of protecting peanuts susceptible to growth of anaflatoxin-producing fungus against contamination by aflatoxin comprisingapplying to the soil where the peanuts are grown a non-aflatoxigenicstrain of Aspergillus parasiticus in an amount effective to inhibitgrowth of native Aspergillus parasiticus strains and their subsequentbiosynthesis of aflatoxin.
 2. A method of claim 1 wherein saidnon-aflatoxigenic strain of Aspergillus parasiticus possesses all of theidentifying characteristics of ARS Culture Collection Number NRRL 18786or NRRL
 18991. 3. A method of claim 1 wherein the non-aflatoxigenicstrain of Aspergillus parasiticus does not produce a detectable amountof dihydrobisfuran-containing intermediate in the aflatoxin biosyntheticpathway.
 4. The method of claim 3 wherein said non-aflatoxigenic strainof Aspergillus parasiticus possesses all of the identifyingcharacteristics of NRRL
 18786. 5. A biocontrol composition for theamelioration of aflatoxin contamination in peanuts comprising a strainof Aspergillus parasiticus having all of the identifying characteristicsof NRRL 18786 or NRRL 18991 and a carrier.